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It is crucial to explore the impact of dietary interventions on depression and develop functional antidepressant foods, due to the significant side effects and poor treatment compliance of antidepressant drugs. Osmanthus fragrans flowers are edible and medicinal materials owing to their delightful floral aroma and significant health benefits. Here, we evaluated the antidepressant effects of the ethanolic extract of O. fragrans var. thunbergii flowers (OFE) and investigated the mechanisms of action on the serotonin system and neuroinflammation, and analyzed the main chemical components of OFE. A single administration of OFE significantly reduced the immobility duration in the forced swimming and tail suspension tests in mice without affecting locomotor activity. OFE exhibited selective enhancing effects on 5‐HTergic function in vivo, as demonstrated by its potentiating effects on 5‐hydroxytryptophan (5‐HTP)‐induced head‐twitch behavior and alleviation effects on reserpine‐induced ptosis deficits. In addition, OFE increased 5‐hydroxytryptamine (5‐HT) concentration and upregulated 5‐HT1A expression in reserpine‐treated mice, further validating its effects on 5‐HT transmission. Interestingly, OFE significantly alleviated microglia activation and the production of inflammatory mediators, both in reserpine‐treated mice in vivo and lipopolysaccharide (LPS)‐stimulated BV‐2 cells in vitro. Additionally, 62 components in OFE were identified using ultra‐performance liquid chromatography quadrupole time‐of‐flight high‐resolution mass spectrometry, and glycoside derivatives were shown to be the major constituents of OFE. The present study showed that OFE can alleviate depressive‐like behaviors by modulating the serotonin system and reducing neuroinflammation. These results suggest that OFE can be valuable dietary supplements with therapeutic effects on depression. [ABSTRACT FROM AUTHOR]